1. Field of the Invention
This invention relates to a colorimetric method and reagent system for the determination of uric acid in biological fluids and more particularly to an improved phosphotungstate method preformed directly on serum and other fluids without deproteinization.
2. Nature and Significance of Accurate Uric Acid Assays
Medical science has long recognized that the measurement of uric acid in blood serum and other body fluids is a very useful and valuable tool in diagnosing and monitoring the course of a variety of pathological conditions. For example, when uric acid is present in abnormally high concentrations in the blood, it tends to crystallize out in the body joints, causing a very painful inflammatory condition known as gout. High uric acid blood levels are also known to be associated with such conditions as uremia and those characterized by an excessive destruction of the nuclei of white blood cells, e.g., leukemia and pneumonia.
In humans, uric acid is the waste product formed from the degradation of purines, which are principally derived from the ingestion of food. In healthy individuals, uric acid is filtered and removed from the blood by the kidneys and excreted in the urine. Determination of the amount or level of uric acid in the urine is thus quite important and useful in diagnosing and evaluating kidney diseases, for a variety of kidney diseases often affect the amount of uric acid excreted. Further, the amount of uric acid in urine provides an index of the amount of purines being metabolized, and thus the comparison of uric acid levels in blood serum and in the urine gives the physician valuable information which is useful in differentiating a variety of diseases affecting purine metabolism and kidney function.
Normal levels or quantities of uric acid in blood serum are very low, generally between only about 0.7 and 7.0 milligrams per 100 milliliters of serum. This very small amount or proportion of uric acid is emphasized by noting that its usual amount is only approximately one-tenth that of other body substances, such as glucose, which are measured for diagnostic purposes.
Accurate measurement of uric acid is demanding and difficult, since there are many substances in blood serum and urine which may be mistaken for uric acid in common assay methods; and if a mistakenly high value for uric acid is reported to the physician, the patient may be erroneously placed on potentially dangerous, expensive, uncomfortable, or unnecessary therapy. For example, if a patient has a high level of serum ascorbic acid, an erroneous and abnormally high uric acid value may be obtained by commonly used uric acid assay methods. This problem is compounded by the fact that even repeated assays to confirm the diagnosis will also produce erroneous results.
In addition to substances normally found in varying degrees in serum which may be mistakenly measured as uric acid, there are a number of substances either ingested in food or which are used as drugs which may interfere with accurate uric acid measurements. For example, caffeine from caffeine-containing beverages such as coffee, tea, and cola drinks, as well as gentisic acid, which is formed in the body from ingestion of aspirin, have been commonly and mistakenly measured as uric acid in prior art assay methods. Vitamin C when taken by the patient greatly elevates serum ascorbic acid levels, and this may also be mistakenly measured as uric acid.
3. Description of Prior Art and Attempts to Improve Specificity
The known methods for the determination of uric acid can be broadly classified into three categories: enzymatic methods utilizing the enzyme uricase, methods based upon the ability of uric acid to reduce alkaline phosphotungstate, and miscellaneous chemical colorimetric methods. The wide variety of methods currently in use today is testament to the fact that none of them is entirely satisfactory, and both past and present research in uric acid methodology has largely been directed toward improving specificity.
As long ago as 1894 (Offer, T. R., Centr. Physiol. 8, p. 801), it was reported that uric acid in an alkaline solution with phosphotungstic acid produces a blue color. Since that time, numerous alterations and modifications have been proposed for the determination of uric acid, all being based upon this ability of uric acid to reduce phosphotungstate. Such phosphotungstate reduction methods have been known and used for decades by thousands of scientists around the world. However, in spite of such longtime use and wide study, these methods still suffer from the fact that they lack specificity in the measurement of uric acid, since substances or chromogens other than uric acid which are found in serum or other body fluids, such as ergothionine, glutathione, ascorbic acid, glucose, creatinine, tyrosine, tryptophan, cystine, cysteine, caffeine, gentistic acid, and a variety of phenolic compounds, also react with alkaline phosphotungstate to give a blue color which may be falsely interpreted as originating from the uric acid chromogen.
Furthermore, in these phosphotungstate methods, protein precipitation and removal is necessary to prevent chromophore formation, and the consequent gross error, from the protein itself, and to prevent the formation of gross turbidity from the interaction of protein with subsequently used reagents. Such manipulation is not only cumbersome, expensive, time-consuming, and requires a large amount of sample, but it also reduces the accuracy of the uric acid assay itself. First, uric acid may be co-precipitated with protein, and thus its level in the sample may be underestimated. Moreover, where dialysis is used for removing protein, uric acid, being a large molecule with affinity for protein, may dialyze more slowly than interfering substances such as ascorbic acid, and thus the effect of the interference may actually be accentuated rather than diminished.
Thus, it can be seen that direct measurement of uric acid in serum without protein removal would be the most efficient and accurate method if interferences could be minimized without having to resort to such laborious and error-prone steps.
Even the phosphotungstate procedures of more recent years have been unable to provide the advantages of the present invention. A brief summary of such procedures helps to illustrate the advantageous significance of the present invention, and it further shows that the present invention is a departure from even phosphotungstate procedures. Moran, in U.S. Pat. No. 3,528,777 (Sept. 15, 1970) added an alkyl sulfate surfactant and hydrazine dihydrochloride in an attempt to eliminate protein interference in a direct phosphotungstate method for detection of uric acid. His method, however, required forty minutes reaction time and still did not provide for elimination of interference from sulfhydryl-containing compounds and other reducing substances in serum. Patel, in U.S. Pat. No. 3,536,448 (Oct. 27, 1970) also used a hydrazine compound, in combination with an amino carboxylic acid sequestering agent, in a phosphotungstate method. Patel's method also required a long reaction time (20 to 30 minutes) and recommended treatment of the sample to remove proteins. Rush, in U.S. Pat. No. 3,649,198 (May 19, 1970) used N-ethylmaleimide to pretreat serum prior to reaction with copper-neocuproine. Presumably, the N-ethylmaleimide reduced sulfhydryl interference, but the long reaction time, the requirement for a serum blank to help correct for other interferences, and the toxic nature of N-ethylmaleimide are serious limitations to this method. Denney, in U.S. Pat. No. 3,801,466 (Apr. 2, 1974) attempted to avoid protein removal and improve specificity for uric acid by treating a sample with uricase to destroy uric acid and then comparing the absorbance of a sample thus treated with the absorbance of an untreated sample following reaction of both samples with alkaline phosphotungstate. Although most common interferences were effectively eliminated by this method of subtracting them out, the problem of interference from endogenous sulfhydryls was not overcome. Furthermore, the procedure is time-consuming and utilizes an enzyme reagent which by nature is of limited stability.